This invention was made with Government support under National Institutes of Health Grant RO1-AI33903. The Government has certain rights in the invention.
The present invention relates to non-human animals which have a natural killer (NK) cell immunodeficiency but have a normal complement of other lymphocytes.
Natural killer (NK) cells were initially described on the basis of their capacity to spontaneously kill certain tumor targets (1). This xe2x80x9cnatural killingxe2x80x9d ability did not require prior deliberate immunization of the host with the tumor cells. This led to the hypothesis that NK cells are involved in tumor surveillance, whereby NK cells eliminate developing cancerous cells before they become established tumors. However, this hypothesis has not been rigorously tested because of the lack of a suitable animal model. Subsequent studies have also suggested that NK cells are involved in other aspects of the immune response. Current data suggest that NK cells have the capacity to respond to certain pathogenic organisms, particularly in the earliest phases (hours to days) of the immune response (2). This contrasts with a more delayed response (days to weeks) in an acquired, specific immune response by B and T cells, that is dependent on clonal expansion and proliferation for specific antibody and cell-mediated responses, respectively (3). On the other hand, NK cells do not appear to require the physical rearrangement of antigen receptor genes, such as such as is required for B (immunoglobulin) and T cell antigen receptors, and are present in mice that lack components of the recombination machinery (4). They must therefore utilize other mechanisms to express a repertoire of receptor molecules that can respond to the universe of tumors or pathogens.
However, NK cells may guide the development of the acquired, specific response, by regulating isotype switching of immunoglobulin isotypes, for example (5). This function appears to be dependent on production of cytokines by the NK cell. Another function attributed to NK cells is their apparent capacity to reject incompatible bone marrow transplants (6). Moreover, NK cells are apparently involved in rejecting solid tissues, such as cardiac allotransplants (7). Thus, NK cells appear to be significant components of the immune system.
All studies of NK cells to date, however, have been limited by the lack of a suitable animal model in which NK cells are selectively and chronically or developmentally absent. The treatment of animals with antibodies which bind to cell surface markers is often employed to deplete cells with those markers in the treated animal. In experiments involving mice, anti-asialoGM1 or anti-NK1.1 antibodies are routinely utilized (8,9). However, those antibodies react with molecules that are expressed on other cells as well as NK cells, such that misleading information can be obtained. For example, a small population of T cells, termed NK/T (or NK1.1+T or NK1 T) cells because they express the NK1.1 antigen otherwise expressed predominantly by NK cells, have been recently shown to be capable of mediating tumor clearance and cytokine production, previously attributed only to NK cells (10). Furthermore, antibody administration is plagued by inherent problems, e.g., that the effect is short lived and that antibody administration may produce other effects on the immune system, such as anti-immunoglobulin formation. Thus, better models for NK cell function in vivo are required.
Immunodeficient hosts can be developed for use as animal models which avoid the problems inherent with the administration of antibodies. Such animals have been used to study various aspects of the immune response. For example, severe combined immunodeficient (scid) mice have a mutation in DNA-dependent protein kinase (DNA-PK) with concomitant abnormalities in the recombination events involved in formation of B and T cell antigen receptors (11). Also, X-linked (xid) mice have a defect in a protein tyrosine kinase which results in a B cell deficiency and nude (nu) mice do not have a thymus and have a defect in T cell development (12). All of these mice have been invaluable in the initial identification and subsequent molecular characterization of B and T cells. As importantly, they have led to an understanding of the contributions of these cells to the normal and deranged immune system. Moreover, these immunodeficient mice have been exploited to carry human cells for the purposes of creating a humanized mouse with which to study infections, tumor formation, drug therapy, and toxicities of various agents (13). On the other hand, these animals have NK cells that can still reject transplanted tissue. Thus, with respect to the elimination of NK cell function, these animals have limitations.
Several mouse strains have been described which contain deficiencies in NK cell function. However, all of these strains also contain other immune deficiencies. These strains have arisen by the mechanisms of spontaneous mutation, gene targeting, or transgene technology.
Spontaneous NK cell deficient mice have been reported but all contain other immune system defects. One of the first described was the beige (Bg) mouse which contains a mutation in a lysosomal transport protein (Lyst) that is not NK cell specific (14). Although this mouse has defects in its capacity to kill tumor targets in vitro, it also has global defects in granule formation, altering the function of other granule-containing lymphocytes. Another mouse, the xe2x80x9cmotheatenxe2x80x9d mouse has a defect in the intracellular tyrosine phosphatase SHP-1 and abnormalities in NK cell development, but also has abnormalities in nearly all hematopoietic lineages (15). Thus, no mouse strain has been reported which has a spontaneous mutation that selectively affects NK cells.
Gene targeting approaches, where the function of a particular gene is eliminated, have resulted in the creation of other mouse strains that have defects in the targeted gene and in NK cells. However, regardless of whether these mice are defective in NK cell function or have a relative absence of NK cells, other aspects of the immune system are invariably and significantly altered due to mutation of the targeted gene (Table 1). Mice with a targeted deficiency in the beta chain of the IL-2 receptor fail to develop NK cells and intestinal epithelial lymphocytes (IEL), and also have defective responses to IL-2 (16). Mice with a targeted deficiency of the common gamma chain of the IL-2 receptor fail to develop T and NK cells (17). Moreover, they are broadly deficient in immune responses because the gamma chain is the signal transduction component of several cytokine receptors, including IL-2, IL-4, IL-7, and IL-15. Mice lacking the Flt-3 molecule have an NK cell deficiency but also have broad defects in hematopoiesis (18). Mice with a targeted deficiency in the Ikaros gene have a defect in lymphopoiesis such that all lymphocytes (B,T, NK cells) fail to develop (19). These mice die in the perinatal period, apparently due to infections. Mice which lack the IRF-1 transcription factor have defects in interferon responses, and hematopoiesis, especially NK, NK/T, and T cell development (20). Moreover, these mice die in the neonatal period. Mice with a targeted deficiency in the Ets-1 transcription factor have an NK deficiency but also have defects in thymic T cell development and T cell antigen receptor-mediated T cell activation (21). Moreover, these mice display an increased perinatal mortality and surviving mice die before adulthood. Mice which lack the Id2 molecule have a defect in NK cell development but still possess some peripheral NK cells with the ability to kill tumor cells (22). Moreover, these mice display an increased neonatal mortality and retarded growth, and fail to develop peripheral lymphoid organs such as lymph nodes and Peyer""s patches. Thus, among gene targeted mice, there are no mice with an absence of NK cells with a relative sparing of other lymphocyte lineages and with an otherwise intact immune system.
Several strains of transgenic mice have been developed which lack NK cells. The Tgxcex526 mouse contains high copy numbers of the human CD3xcex5 gene (23). Although it lacks NK cells, it also lacks T cells. An IL-2 receptor xcex2 chain transgenic mouse lacks NK cells but also lacks epidermal T cells (24). In addition, T cells in those transgenic mice are hyper-responsive to IL-2. The granzyme A-diptheria toxin mouse lacks a substantial number of NK cells but is also devoid of T cell populations (25). Thus, no transgenic mice with a selective deficiency of NK cells has been reported.
Another strategy to create an NK cell deficient mouse is to generate mice with abnormalities in NK cell effector mechanisms such that the NK cells are present but functionally impotent. Perforin-deficient and granzyme-deficient mice contain NK cells, but these NK cells have deficiencies because perforin and granzymes are normally contained in NK cell granules that are released upon activation to mediate killing of cellular targets (26,27). However, because these components are also required for the cytolytic activity of cytotoxic T lymphocytes (CTL), the CTLs are also defective. Mice with defects in the Fcxcex3RIII molecule contain NK cells but fail to develop antibody-dependent cellular cytotoxicity (ADCC) because this molecule is utilized by the NK cell for this response (28). However, natural killing is intact and other cells that normally express this receptor also fail to generate ADCC function. IL-18 defective mice have abnormal NK cell responses although the number of NK cells is apparently normal (29). Thus, mice with defects in NK cell effector function also have other defects. On the other hand, NK cells present in these mice presumably possess normal NK cell effector functions such as interferon-y production, other than those disrupted effector functions.
Therefore, no animal species has been reported which has either an absolute or functional deficiency in NK cells with relative sparing of other immune system components. The development of such an animal, such as the mouse disclosed herein, represents a significant advance for the scientific fields of immunology, cancer, hematology, transplantation, and infectious diseases.
With regard to xenogeneic transplantation, host immune systems are involved in rejecting transplanted tissues. Thus far, it has been possible to transplant many human tissues into animals that lack B and T cells. This has obvious utility in that the animals can then be infected with pathogens that infect the implanted human tissues or human tumors, permitting testing of new therapies or virulence factors (30, 31). However, not every human tissue can apparently be transplanted into the B and T cell deficient animals, perhaps reflecting the capacity of NK cells to reject transplants (13). Moreover, injection of antibodies that react with NK cells result in enhanced xenogeneic transplant survival, implying a role for NK cells in transplant rejection. The development of animal models that lack B, T, and NK cells, yet which are viable, reproductive animals that reach adulthood will be useful in further investigation.
Among the several objects of the invention, therefore, may be noted the provision of a transgenic mammal in which there is a substantial deficiency of NK cells with normal immunoglobulin levels and normal numbers of T and B lymphocytes in peripheral blood and spleen. Also provided are transgenic mammals with combined deficiencies in NK along with T and/or B cells, produced by mating the NK deficient transgenic mammal with mammals deficient in T and/or B cells. These mammals are useful for studying the immunological role of NK, T, and B cells and as animal models for human diseases such as immunodeficiency diseases or cancer.
A further object of the invention is the provision of non-human mammals which do not reject or have a substantially diminished capacity to reject transplants of human tissues. Such mammals are useful for assessing, for example, the toxicity, carcinogenicity, or therapeutic effect of chemical compositions on the transplanted human tissue. They can also help identify disease agents.
Another object of the invention is the provision of non-human mammals with substantially diminished ability to kill or reject cancer tumors. These mammals are useful for studying cancer and for developing cancer therapies. They should also have increased susceptibility to carcinogenic factors and are thus useful to assess the carcinogenic potential of administered agents.
Therefore, the present invention is directed to a non-human mammal genetically having:
a) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S. or B16 tumor cells; and
b) a substantially normal complement of other lymphocytes.
The present invention is further directed to a non-human mammal which comprises a transgene which has a granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed.
The invention is also directed to a method of producing a transgenic non-human mammal which has a deficiency in natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells. The method comprises the following steps:
a) introducing a transgene into an embryonal cell of the mammal, the transgene comprising a granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed; and
b) identifying a mammal derived from the embryonal cell which contains the stably integrated transgene and has a deficiency of natural killer cells.
The invention is also directed to a non-human mammal which comprises i) a granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed, and ii) a substantial deficiency in T cells.
The present invention is further directed to a method of producing a progeny non-human mammal which has: i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, and ii) a substantial deficiency in T cells. The method comprises the following steps:
a) mating a first non-human mammal with a second non-human mammal, wherein the first non-human mammal has the deficiency of natural killer activity and a substantially normal complement of other lymphocytes, and wherein the second non-human mammal has a substantial deficiency in T cells and a substantially normal complement of other lymphocytes; and
b) selecting progeny derived from the mating of step a) which has a deficiency of natural killer cells and T cells.
The present invention is also directed to a non-human mammal produced by the method of described immediately above.
The invention is further directed to a non-human mammal which comprises i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, and ii) a substantial deficiency in B cells.
The invention is also directed to a non-human mammal which comprises i) a granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed, and ii) a substantial deficiency in B cells.
The invention is still further directed to a method of producing a progeny non-human mammal which has i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, and ii) a substantial deficiency in B cells. The method comprises the following steps:
a) mating a first non-human mammal with a second non-human mammal, wherein the first non-human mammal has the deficiency of natural killer activity and a substantially normal complement of other lymphocytes, and wherein the second non-human mammal has a substantial deficiency in B cells and a substantially normal complement of other lymphocytes; and
b) selecting progeny derived from the mating of step a) which has a deficiency of natural killer cells and B cells.
The present invention is also directed to a non-human mammal produced by the method described immediately above.
The present invention is further directed to a non-human adult mammal which comprises i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, ii) a deficiency of T cells, and iii) a deficiency of B cells.
The present invention is still further directed to a non-human mammal which comprises i) a granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed, ii) a substantial deficiency in T cells, and iii) a substantial deficiency in B cells.
The invention is also directed to a method of producing a progeny non-human mammal which has i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, ii) a deficiency of T cells, and iii) a deficiency of B cells. The method comprises the following steps:
a) mating a first non-human mammal with a second non-human mammal, wherein the first non-human mammal has the deficiency of natural killer activity and a substantially normal complement of other lymphocytes, and wherein the second non-human mammal has a substantial deficiency in T cells and B cells; and
b) selecting progeny derived from the mating of step a) which are deficient in natural killer cells, T cells, and B cells.
The present invention is also directed to a non-human mammal produced by the method described immediately above.
The invention is further directed to a method of producing a non-human mammal which has i) a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, ii) a deficiency of T cells, and iii) a deficiency of B cells. The method comprises the following steps:
a) directing a first mating of a first non-human mammal with a second non-human mammal, then directing a second mating of the progeny derived from the first mating with a third non-human mammal, wherein one of the non-human mammals has the deficiency of natural killer activity and a substantially normal complement of other lymphocytes, wherein another of the non-human mammals has a substantial deficiency in T cells and a substantially normal complement of other lymphocytes, and wherein the remaining nonhuman mammal has a substantial deficiency in B cells and a substantially normal complement of other lymphocytes; and
b) selecting progeny derived from the second mating which has a deficiency in natural killer cells, T cells and B cells.
The present invention is also directed to a non-human mammal produced by the method described immediately above.
In a further embodiment, the present invention is directed to a method for producing an animal containing human tissues. The method comprises the following steps:
a) providing a non-human mammal which has a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, wherein the non-human mammal comprises a transgene comprising a mouse granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed; and
b) transplanting human tissue into the non-human mammal.
The invention is further directed to a method for evaluating the effects of a composition on human tissue. The method comprises the following steps:
a) providing a non-human mammal which has a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, wherein the non-human mammal comprises a transgene comprising a mouse granzyme A gene wherein a Ly49A gene is inserted into the granzyme A gene such that the Ly49A gene is expressed; and
b) contacting the composition with the human tissue in the animal.
Additionally, the present invention is directed to a method for determining whether a human disease is caused by a pathogenic agent. The method comprises the following steps:
a) providing a non-human mammal which has a deficiency of natural killer activity of at least 50% as measured by reduction in specific lysis of YAC-1, RMA-S, or B16 tumor cells, wherein the non-human mammal comprises a transgene comprising a mouse granzyme A gene wherein a mouse Ly49A gene replaces the start codon of the mouse granzyme A gene;
b) transplanting non-diseased human tissue into the non-human mammal;
c) contacting diseased human tissue with the non-diseased human tissue;
d) determining whether the non-diseased human tissue acquires the disease.
In an additional embodiment, the invention is directed to a method of studying natural killer cells. The method comprises the following steps:
a) obtaining a NKDef mouse and a mouse having wild-type natural killer activity;
b) infecting the NKDef mouse and the mouse having wild-type natural killer activity with a pathogen or with cancer cells; and
c) evaluating differences in immunity between the NKDef mouse and the mouse having wild-type natural killer activity.
The present invention is also directed to mouse natural killer cells having a deficiency of interferon-xcex3 production of at least 50% when compared to wild-type mouse natural killer cells.
The present invention is additionally directed to a method of studying natural killer cells. The method comprises comparing natural killer cells from an NKDef mouse with natural killer cells from a mouse having wild-type natural killer activity.
Other features, objects and advantages of the present invention will be in part apparent to those skilled in the art and in part pointed out hereinafter. All references cited in the instant specification are incorporated by reference. Moreover, as the patent and non-patent literature relating to the subject matter disclosed and/or claimed herein is substantial, many relevant references are available to a skilled artisan that will provide further instruction with respect to such subject matter.